Cell chamber for chemotaxis assay

ABSTRACT

A chemotaxis assay instrument comprising a plate assembly with a bottom plate, a gasket seal mounted to the bottom plate and an intermediate plate mounted on the other side of the gasket seal and spaced by the gasket seal from the bottom plate. The intermediate plate defines a plurality of wells to hold a sample and a filter membrane is mounted on the intermediate plate. A second intermediate plate is spaced from the first intermediate plate by the filter membrane and second gasket seal is mounted on the other side of the second intermediate plate with a top plate mounted on said second gasket seal and spaced by the second gasket seal from the second intermediate plate, the plate assembly being held together by fasteners in a sealed relationship.

BACKGROUND OF THE INVENTION

Mammalian cells are usually cultured in glass or plastic vessels, eitherin suspension or as an attached layer, completely surrounded by culturemedia. Pseudopodia protrusion is a prominent feature of mammalian cellsboth invitro and invivo. Through their surfaces, cells can interact andsense the changes in the outside world. In studies of model membranesystems, steps in the fusion of membrane have been identified. In orderfor the fusion process to take place, certain requirements have to befulfilled. Initially the membranes have to be close enough to makecontact. This is generally prevented by large membrane glycoproteinswhich sterilly hinder the membranes preventing a close enough contact toallow fusion. Because of the charged surface of the membranes, they mayrepel, thereby preventing any closer contact. Therefore, there must besome mechanism within the cell to control which membrane will undergofusion. This may be regulated by specific fusion proteins similar to theones isolated from the influenzia and sendia virus.

The use of chambers for cell culture and chemotaxis assay is known inthe prior art.

U.S. Pat. No. 3,821,087 discloses an apparatus having semi-permeabletube shaped membranes allowing the growth of cells invitro. Cells areallowed to settle on the outside surfaces of the membranes in a nutrientmedium environment while the membranes are continuously profused withoxygenated nutrient medium flowing through the membranes. The nutrientsubstances diffuse from the perfusing medium through the membrane walland into the cells, while cell products diffuse from the cell throughthe membrane wall into the perfusate from which the cell's products maybe recovered.

U.S. Pat. Nos. 4,087,327 and 4,201,845 are directed to a cell culturereactor used in the growth of cells invitro which employs elongateselectively permeable hollow fibers arranged in a shallow layerconfiguration as a matrix for cell attachment on the outer surface ofthe fibers. The flow path of the culture medium is directed by adistributor plate which serves as a manifold to provide uniformdistribution of the medium through the fibers and a flow path which isupward and transverse to the plane of the elongate axis of the fibers.

U.S. Pat. No. 4,228,243 discloses an apparatus and method forpropagating tissue cultures in a plurality of stacked frames and plateswhich are clamped together. The plates are stacked on resilient gasketswhich are provided to stop leakage between the uppermost dish andcoverplates and overflow passages are provided in the plates throughwhich the media, cell suspensions and gases can pass.

U.S. Pat. No. 4,299,920 discloses a receptacle for cell culturesconstructed of a base plate with a wall assembly defining a plurality ofchambers detachably, adhesively joined to the base plate. The portion ofthe wall assembly adjacent to the base plate consists of a noncytotoxicelastomeric synthetic material which adheres to the base plate in aliquid-tight manner. On completion of cultivation of the cells the baseplate can be manually separated from the wall section.

U.S. Pat. No. 4,304,865 discloses an apparatus having a harvester platewith a plurality of wells recessed into one surface. A disc-shaped sheetof filter paper placed over the top surface of the harvester plate and amodified Terasaki plate with an outer wall section and conically shapedwells recessed into its surface is mounted over the filter paper andharvester plate. When the plate is maintained in an upside downorientation the material is transferred directly to the filter paperdisc.

U.S. Pat. No. 4,324,859 discloses a microbiological container with asubstrate having insertable elements. The lower edge of each elementforms a sealed relationship with the bottom of the container formingseparate regions with isolated substrate portions. A cover can be placedon the insertable element in order to cover the respective isolatedsection.

U.S. Pat. No. 4,326,028 discloses a cylindrical dish with twocylindrical compartments divided by a perforated jell support structurewith a temporary seal. After the microorganisms are grown in compartmentA, the dish is inverted, the seal removed, and a second nutriate mediumpoured into compartment B.

U.S. Pat. No. 4,634,676 discloses a replica plating device comprising acontainer which holds cells and a skirted press member which fits intothe container. Attached to the exterior bottom surface of the bottomwall of the press member is a layer of compressible material such assoft expanded polyurethane or hard polyurethane foam. The compressiblematerial serves to cushion the pressing effect of the replica platingdevice against the cells and culture medium during use.

U.S. Pat. No. 4,661,458 discloses a device used for the culture of cellsincorporating several modules of different functions for regulating thecell growth environment, providing a suitable cell growth substrate orseparating the desired product from interfering substances. Each moduleis constructed of a series of membranes separated by a solid supportmaterial which is channeled to provide a series of parallel capillariesfor the flow of fluids.

U.S. Pat. No. 4,720,462 discloses a culture tube divided into fivesealed chambers with cell impermeable hydrophilic tubes extending fromone chamber to another chamber.

A cell permeable filter tube extends along the initial horizontal axisof the straight culture tube from one end of the culture chamber to theother. The filter tube has an average porosity over 100 microns,preferably between about 100 and 500 microns such that under normaloperating conditions of cell sloughage the tube does not occlude. Cellsare forced under pressure into the tube and through the wall of the cellpermeable tube.

U.S. Pat. No. 4,748,124 discloses a closed cell culture device usingspaced sheets of gas permeable, liquid impermeable material with a thirdsheet sandwiched between the first and second spaced sheets which isselectively permeable to a class of molecules having a molecular weightranging from 8,000 to 15,000 molecules. All of the sheets are held in ahousing chamber formed by four concentric ring members, the first andthird sheets define a first closed compartment and the second and thirdsheets define a second enclosed compartment with each compartment havingan access port. The rings are sealed together in compression byattachment screws.

The use of a small diameter pore filter which is small enough to preventwhole cell migration in the chemotaxis assay allows the cells to firstextend their pseudopodia to a lower compartment while the cell nucleiremain in the upper compartment. Consequently the separation between thetwo compartments by mono layered cells anchored to the filter throughtheir extended pseudopodia permits the collection of extendedpseudopodia as well as substances secreted into the lower walls. Thus,depending on the pore size of the filters, the pseudopodia chamber canbe used for the study of either whole cell migration or cell pseudopodiaprotrusion. Although cell pseudopodia is not new to researchers studyingcell movement, no one has previously been able to isolate these cellularprocesses. Furthermore, in order to study the morphological changesinvolved in the intermediate steps between triggering the chemotaxticsignal and the end result i.e. cell locomotion, one had to rely on acomplicated technique such as time lapse cinemotagraphy. The results ofsuch cinemotagraphy are difficult to interpret as well as uncertain.

One prior art assay for enutorphoril chemotaxis which ha been widelyused for studying cell movement is the Boyden chamber assay which isshown in FIGS. 1 and 1A. The Boyden chamber is composed of two partswhich constitute an upper and a blind lower well. Chemo attractants areadded to the lower well before placing the membrane which separates thelower from the upper well. In this assay the conditions in the lowerwell cannot be changed once the experiment has been started. A secondprior art chamber which has been used for chemotaxis assay is shown inFIGS. 2 and 2A. This chamber is composed of a bottom plate with a singleor multiple blind wells, a filter and a top plate with matching diameterwells which is attached to the bottom plates by a series of bolts. Incontradistinction to this device, the newly designed inventive chamberprovides free access to both the upper as well as the lower wellsthroughout the experiment. This permits the study of isolated or intactpseudopodial protrusions independent from their cell bodies. Moreover,since the cell monolayer can physically separate the two compartments inthe new chamber, one can further study the changes in cell responses todifferent legans after changing the conditions in either the upper orlower wells or both.

SUMMARY OF THE INVENTION

A chemotaxis assay instrument comprising a bottom plate, a gasket filtermounted to said bottom plate surface and, an intermediate plate defininga plurality of wells to hold a sample mounted on the other side of saidgasket filter, form one compartment of the instrument. A secondintermediate plate is spaced from the first intermediate plate by apolycarbonate membrane filter having a pore size ranging from 3 to 8microns. A top plate is mounted to the first intermediate plate with agasket filter positioned between the top plate and second intermediateplate to form a second compartment of the instrument. Bolts hold theplates and filter membrane together in a sealed relationship.

It is an object of the present invention to provide an apparatus for thesimple, economical and effective culture of cells.

It is a further object of the present invention to provide for a meansof collecting and/or removing cell pseudopodia.

It is yet another object of the present invention to allow free accessto upper and lower wells at any time during the assay without disturbingthe cell pallet.

These and other objects and advantages of the present inventiveapparatus will become more readily apparent in the following detaileddescription thereof together with the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 1A are respectively top plan and exploded cross sectionalviews of the prior art Boyden chamber;

FIGS. 2 and 2A are top plan and exploded cross section views of anotherprior art chemotaxis chamber;

FIG. 3 is a top plan view of a preferred embodiment of the invention;and

FIG. 4 is an exploded cross sectional view of the preferred inventivechamber embodiment as viewed across line 4'--4' of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

The preferred embodiment and best mode of the invention is shown byFIGS. 3 and 4. Representative prior art is shown by FIGS. 1 and 2. InFIGS. 3 and 4 the inventive chemotaxis chamber assembly 10 is afour-tiered instrument made of clear acrylic with two smooth surfacedmiddle plates 12 and 14 provided with respective threaded throughgoingapertures 13 and 15 and two additional clear acrylic plates forming thetop plate 16 and bottom plate 18. It should be noted that any suitablematerial could be used as the material of the plates but the same arepreferably of a clear and an inert material.

One unique feature of the present invention is the use of a double setof stainless steel hardware comprising a suitable number of bolts whichwould provide effective sealing between the plates, filter and gaskets.One set of six stainless steel bolts 20 having a head 21 with a threadedportion 23 adjacent to the head and a threaded portion 23a remote fromthe threaded portion 23 distal from head 21 enters the assembly throughthroughgoing holes in the bottom plate 18 and with the gasket 22threadably seals the bottom plate 18 to the lower middle plate 14 viathe engagement of threaded portion 23 into threaded aperture 15. Asecond set of stainless steel bolts 24 having a head 21 with a threadedportion 25 adjacent to the head and a threaded portion 25a remote fromthe threaded portion 25 and distal from head 21 enters surface 17 andthe associated bolt seat 27 of the top plate 16 into throughgoing holesin the top plate 16 and likewise pinches a gasket 26 between the topplate 16 and the threadably attached upper middle plate 12. The threadedportion 25 of bolt 24 is threaded in the threaded aperture 13 of theupper middle plate 12, holding plate 16, gasket 26 and upper middle 12in a fixed secured relationship. The bottom plate 18, lower middle plate14 and associated gaskets are removably held in place by thumb nuts orstandard nuts 32 fixedly attached to distal threaded portion 25a. It isof course apparent that compartment assemblies work in the same manner.

While the present embodiment uses threaded bolts, any other suitabletypes of securing means known in the prior art which accomplish the samesealing results and sectionality of a compartment could be used. Withthe hardware thus installed, the bottom/lower middle plate group 18, 14and top/upper middle plate group 16, 12 form two assay sections withclosed or blind wells 28 numbering 48 separate wells. Each set of sixhardware bolts 20, 24 is not positioned in the same hole pattern.Therefore, in the top/upper middle plate group 16, 12 bolts 24 arescrewed in threaded aperture 13 of plate 12 and penetrate an unoccupiedset of holes in the bottom/middle plate group 18/14 and vice versa. Theupper and lower plate groups are joined with an interposed gasket andfilter 30 of polycarbonate or cellulose nitrate to form 48 sealedchambers or blind wells 28 of 50 μl each. It is apparent that thechambers or wells could vary in number from the number set forth; eitherbeing greater or lesser as desired. The filter is preferably 25×80 mmand constructed of a polycarbonate membrane having a pore size rangingfrom 1 to 8 microns diameter. The filter can also be coated with TYPE 1collagen, TYPE IV collagen, fibronectin or laminin if desired. It isalso envisoned that the filters could be constructed of any suitableporous material.

The chambers are designed so that the thumb nuts 32 mounted in recesses34 on surfaces 17 and 19 tie the two plate sets together and do not bearon both the top and bottom plates 16 and 18. This allows each set of sixbolts with associated thumb nuts installed to join the middle lateeither to the bottom or top pair. Consequently either the top or bottomplate can be removed without disturbing the seal between the two middleplates 12, 14 and the filter 30. This permits free access to the upperwells (cell compartment) 40 and lower wells (pseudopodia compartment) 50without disturbing the cell plate at any time during the assay. With afilter and gasket 22 and 26 interposed between the top and bottomsections, the fully assembled instrument creates 48 closed wellsseparated by a filter.

It is further envisioned that the plates could be sectioned so that onlya portion of the cells on a particular side are exposed.

An experiment is started by filling the 48 wells in the assembled bottomsection 50. A filter 30 can then be placed over the filled wells and theupper middle plate 12, attached with thumb nuts. The assay instrumentcan then be incubated without the top plate 16 attached. Afterincubation the top plate 16 is attached, the chamber inverted and thebottom plate 18 removed without breaking the seal of the filter 30between the middle plates 12 and 14. This allows access to the bottomwells on the bottom side of the filter where cells or cell pseudopodsmay be studied or fluid sampled or replaced. Thus it can be seen thatthe hardware is designed so that either the top plate or the bottomplate can be removed without disturbing the seal between the three otherplates.

While the general embodiments of the present invention have beendescribed, it will be apparent to those of ordinary skill in the artthat various alternative configurations and embodiments can readily beadapted to the present invention and are considered to fall within thescope thereof as set forth in the following claims.

What we claim is:
 1. A chemotaxis assay instrument comprising a bottomplate, seal means mounted to said bottom plate, an intermediate platemounted on the other side of said seal means and spaced by said sealmeans from said bottom plate, said intermediate plate defining aplurality of wells to hold a sample, a filter mounted to saidintermediate plate, a second intermediate plate spaced from said firstintermediate plate by said filter, second seal means mounted to saidsecond intermediate plate, a top plate mounted to said second seal meansspaced by said second seal means from said second intermediate plate,and means to hold said plates together in a sealed relationship.
 2. Aninstrument as claimed in claim 1 wherein said holding means comprises aplurality of bolts.
 3. An instrument as claimed in claim 2 wherein saidbolts are positioned on said top plate and said bottom plate end areoffset from each other.
 4. An instrument as claimed in claim 1 whereinsaid top, bottom, and intermediate plates are constructed of clearacrylic plastic.
 5. An instrument as claimed in claim 2 wherein saidbolts are constructed of stainless steel.
 6. An instrument as claimed inclaim 1 wherein said seal means are polycarbonate membranes.
 7. Aninstrument as claimed in claim 1 wherein said filter is constructed ofpolycarbonate.
 8. An instrument as claimed in claim 1 wherein saidplates are attached together with thumbnuts.
 9. An instrument as claimedin claim 1 wherein said assembled instrument creates two separatedcompartments, each of which has a plurality of closed wells separated bya filter.
 10. An instrument as claimed in claim 9 wherein said closedwells are at least 48 in number.
 11. An instrument as claimed in claim 1wherein said instrument forms a plurality of wells having a cellcompartment and a pseudopodia compartment.
 12. An instrument as claimedin claim 1 wherein said intermediate plates define a plurality of wells.13. An instrument as claimed in claim 1 wherein said filter is apolycarbonate membrane with a pore size ranging from 1 to 8 microns. 14.An instrument as claimed in claim 1 wherein said filter is constructedof polycarbonate coated with TYPE I collagen.
 15. An instrument asclaimed in claim 1 wherein said filter is constructed of polycarbonatecoated with TYPE II collagen.
 16. An instrument as claimed in claim 1wherein said filter is constructed of polycarbonate coated withfibronectin.
 17. An instrument as claimed in claim 1 wherein said filteris constructed of polycarbonate coated with laminin.
 18. A cell cultureapparatus for cell assay comprising a plurality of cell-impermeableplates, at least two plates defining a plurality of cell culture wellsseparated by a filter membrane of porous construction having a pore sizesuitable to allow the pseudopods of cells to pass therethrough withoutallowing the nucleus of the cell to pass therethrough, outer platesspaced from the outer surface of each of said two plates by a filtermember between each outer plate and the adjacent inner plate and lockingmeans holding said plates in a fixed sealed relationship.
 19. A cellculture apparatus as claimed in claim 18 wherein said locking meanscomprises a plurality of fasteners mounted to one outer plate and atleast its adjacent spaced plate and a second plurality of fastenersmounted to the other outer plate and at least its adjacent spaced platepositioned offset from said first plurality of fasteners.
 20. A cellculture apparatus as claimed in claim 18 wherein said filter membranehas throughgoing pores ranging from 1-8 microns in size.
 21. A cellculture apparatus as claimed in claim 18 wherein said plates areconstructed of a clear plastic.
 22. A selective access dual compartmentcell culture assay device comprising two plates defining a plurality ofcell culture wells separated by a membrane having a pore size rangingfrom 2-8 microns, at least one sheet of gas permeable, liquidimpermeable material mounted to the opposite side of each of saidplates, an end plate mounted opposite each plate and spaced from saidplate by said sheet of gas permeable, liquid impermeable material andlocking means securing at least said one plate and its respectiveopposing end plate together to form a first compartment and said otherplate and its respective opposing end plate together to form a secondcompartment and means to hold all of said plates together in a fixedsealed relationship.